RESUMEN
Human hematopoietic stem cells (HSCs), like their counterparts in mice, comprise a functionally and molecularly heterogeneous population of cells throughout life that collectively maintain required outputs of mature blood cells under homeostatic conditions. In both species, an early developmental change in the HSC population involves a postnatal switch from a state in which most of these cells exist in a rapidly cycling state and maintain a high self-renewal potential to a state in which the majority of cells are in a quiescent state with an overall reduced self-renewal potential. However, despite the well-established growth factor dependence of HSC proliferation, whether and how this mechanism of HSC regulation might be affected by aging has remained poorly understood. To address this knowledge gap, we isolated highly HSC-enriched CD34+CD38-CD45RA-CD90+CD49f+ (CD49f+) cells from cord blood, adult bone marrow, and mobilized peripheral blood samples obtained from normal humans spanning 7 decades of age and then measured their functional and molecular responses to growth factor stimulation in vitro and their regenerative activity in vivo in mice that had undergone transplantation. Initial experiments revealed that advancing donor age was accompanied by a significant and progressively delayed proliferative response but not the altered mature cell outputs seen in normal older individuals. Importantly, subsequent dose-response analyses revealed an age-associated reduction in the growth factor-stimulated proliferation of CD49f+ cells mediated by reduced activation of AKT and altered cell cycle entry and progression. These findings identify a new intrinsic, pervasive, and progressive aging-related alteration in the biological and signaling mechanisms required to drive the proliferation of very primitive, normal human hematopoietic cells.
Asunto(s)
Células Madre Hematopoyéticas , Mitógenos , Adulto , Humanos , Animales , Ratones , Integrina alfa6/metabolismo , Mitógenos/metabolismo , Células Madre Hematopoyéticas/metabolismo , Antígenos CD34/metabolismo , Proliferación Celular , Puntos de Control del Ciclo Celular , Ciclo Celular , Péptidos y Proteínas de Señalización Intercelular/metabolismoRESUMEN
Recent advances in single-cell molecular analytical methods and clonal growth assays are enabling more refined models of human hematopoietic lineage restriction processes to be conceptualized. Here, we report the results of integrating single-cell proteome measurements with clonally determined lymphoid, neutrophilic/monocytic, and/or erythroid progeny outputs from >1000 index-sorted CD34+ human cord blood cells in short-term cultures with and without stromal cells. Surface phenotypes of functionally examined cells were individually mapped onto a molecular landscape of the entire CD34+ compartment constructed from single-cell mass cytometric measurements of 14 cell surface markers, 20 signaling/cell cycle proteins, and 6 transcription factors in â¼300 000 cells. This analysis showed that conventionally defined subsets of CD34+ cord blood cells are heterogeneous in their functional properties, transcription factor content, and signaling activities. Importantly, this molecular heterogeneity was reduced but not eliminated in phenotypes that were found to display highly restricted lineage outputs. Integration of the complete proteomic and functional data sets obtained revealed a continuous probabilistic topology of change that includes a multiplicity of lineage restriction trajectories. Each of these reflects progressive but variable changes in the levels of specific signaling intermediates and transcription factors but shared features of decreasing quiescence. Taken together, our results suggest a model in which increasingly narrowed hematopoietic output capabilities in neonatal CD34+ cord blood cells are determined by a history of external stimulation in combination with innately programmed cell state changes.
Asunto(s)
Antígenos CD34/metabolismo , Linaje de la Célula , Sangre Fetal/metabolismo , Células Madre Hematopoyéticas/metabolismo , Proteoma/análisis , Análisis de la Célula Individual/métodos , Diferenciación Celular , Células Cultivadas , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Humanos , Proteoma/metabolismoRESUMEN
A series of 2'-hydroxychalcone derivatives was synthesized and the effects of all the compounds on growth of A549 lung cancer cell were investigated. The results showed that all compounds had inhibitory effects on the growth of A549 lung cancer cells and compound possessed the highest growth inhibitory effect and induced autophagy of A549 lung cancer cells.
Asunto(s)
Autofagia/efectos de los fármacos , Chalconas/farmacología , Descubrimiento de Drogas , Neoplasias Pulmonares/patología , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Chalconas/síntesis química , Chalconas/química , Humanos , Concentración 50 Inhibidora , L-Lactato Deshidrogenasa/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , NecrosisRESUMEN
We develop a pyrazoline-based fluorescent sensor for biological Zn(2+) detection. The sensor shows good binding selectivity for Zn(2+) over competing metal with 40-fold fluorescence enhancement in response to Zn(2+). The new probe is cell-permeable and can be used to detect intracellular zinc ions in living neuron cells.
Asunto(s)
Colorantes Fluorescentes/química , Neuronas/química , Pirazoles/química , Zinc/análisis , Animales , Colorantes Fluorescentes/síntesis química , Estructura Molecular , Neuronas/citología , Células PC12 , Pirazoles/síntesis química , RatasRESUMEN
CO, a gas molecule that is harmful to living organisms, has a high affinity with hemoglobin, which will cause severe hypoxia. However, in recent years, researchers have discovered that endogenous CO, similar to NO, is one of the messenger molecules, which has a certain regulatory effect in many physiological and pathological processes in the respiratory system, cardiovascular system, and nervous system. Therefore, it is urgent to explore an effective method to monitor the role of CO under physiological and pathological conditions. Herein, we designed and synthesized a near-infrared small-molecule fluorescent probe for the detection of CO in living cells. In this design, a two-site BODIPY dye was introduced as the fluorophore, and the allyl chloroformate part as the CO reactive group. The probe displays excellent sensitivity, selectivity, and a good linear relationship to CO. Furthermore, it shows good biocompatibility and low cytotoxicity. This probe has been successfully applied to the detection of CO in a variety of cells. The developed fluorescent probe can serve as a potential molecular imaging tool for in vivo imaging and detection of CO.
RESUMEN
Hypochlorous acid (HOCl), a highly potent oxidant of reactive oxygen species, plays critical roles in many physiological and pathological processes. In this work, a novel coumarin-based fluorescent probe, Cou-HOCl, was prepared for the detection of HOCl. The probe exhibited good selectivity over other analytes, excellent sensitivity with a detection limit of 16 nM, and fast response within 5 s. And further study demonstrated that the probe could be used not only to image exogenous HOCl in various cells, but also to determine the fluctuating levels of HOCl in macrophage cells during inflammation.
RESUMEN
Elucidation of the identity and diversity of mechanisms that sustain long-term human blood cell production remains an important challenge. Previous studies indicate that, in adult mice, this property is vested in cells identified uniquely by their ability to clonally regenerate detectable, albeit highly variable levels and types, of mature blood cells in serially transplanted recipients. From a multi-parameter analysis of the molecular features of very primitive human cord blood cells that display long-term cell outputs in vitro and in immunodeficient mice, we identified a prospectively separable CD33+CD34+CD38-CD45RA-CD90+CD49f+ phenotype with serially transplantable, but diverse, cell output profiles. Single-cell measurements of the mitogenic response, and the transcriptional, DNA methylation and 40-protein content of this and closely related phenotypes revealed subtle but consistent differences both within and between each subset. These results suggest that multiple regulatory mechanisms combine to maintain different cell output activities of human blood cell precursors with high regenerative potential.
Asunto(s)
Proliferación Celular , Separación Celular/métodos , Sangre Fetal/citología , Mitosis , Lectina 3 Similar a Ig de Unión al Ácido Siálico/metabolismo , Análisis de la Célula Individual/métodos , Células Madre/metabolismo , Animales , Biomarcadores/metabolismo , Trasplante de Células Madre de Sangre del Cordón Umbilical , Metilación de ADN , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genotipo , Humanos , Masculino , Ratones Transgénicos , Fenotipo , Factores de Tiempo , TranscriptomaRESUMEN
We designed and synthesized a new pyrazoline-based turn-on fluorescence probe for Zn(2+) by the reaction of chalcone and thiosemicarbazide. The structure of the probe was characterized by IR, NMR and HRMS spectroscopy. The probe (L) exhibits high selectivity and sensitivity for detecting Zn(2+) in buffered EtOH/HEPES solution (EtOH/HEPES=1/1, pH 7.2) with 80-fold fluorescence enhancement, which is superior to previous reports. Job's plot analysis revealed 1:1 stoichiometry between probe L and Zn(2+) ions. The association constant estimated by the Benesi-Hildebrand method and the detection limit were 3.92×10(3)M(-1) and 5.2×10(-7)M, respectively. A proposed binding mode was confirmed by (1)H NMR titration experiments and density functional theory (DFT) calculations. The probe is cell-permeable and stable at the physiological pH range in biological systems. Because of its fast response to Zn(2+), the probe can monitor Zn(2+) in living cells. Moreover, the selective binding of L and Zn(2+) was reversible with the addition of EDTA in buffered EtOH/HEPES solution and Zn(2+) could be imaged in SH-SY5Y neuron cells.